Recurrent symbiont recruitment from fungal parasitesin cicadasYu Matsuuraa,b,1, Minoru Moriyamab, Piotr Łukasikc, Dan Vanderpoolc, Masahiko Tanahashib,d, Xian-Ying Mengb,John P. McCutcheonc, and Takema Fukatsub,e,f,1

aTropical Biosphere Research Center, University of the Ryukyus, 903-0213 Nishihara, Japan; bBioproduction Research Institute, National Institute ofAdvanced Industrial Science and Technology, 305-8566 Tsukuba, Japan; cDivision of Biological Sciences, University of Montana, Missoula, MT 59812;dDepartment of Applied Chemistry, National Chiao Tung University, 30010 Hsinchu, Taiwan; eDepartment of Biological Sciences, Graduate School ofScience, University of Tokyo, 113-0033 Tokyo, Japan; and fGraduate School of Life and Environmental Sciences, University of Tsukuba, 305-8572Tsukuba, Japan

Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved May 10, 2018 (received for review February 23, 2018)

Diverse insects are associated with ancient bacterial symbionts,whose genomes have often suffered drastic reduction and de-generation. In extreme cases, such symbiont genomes seem almostunable to sustain the basic cellular functioning, which comprises anopen question in the evolution of symbiosis. Here, we report an insectgroup wherein an ancient symbiont lineage suffering massivegenome erosion has experienced recurrent extinction and replace-ment by host-associated pathogenic microbes. Cicadas are associatedwith the ancient bacterial co-obligate symbionts Sulcia and Hodgkinia,whose streamlined genomes are specialized for synthesizing es-sential amino acids, thereby enabling the host to live on plant sap.However, our inspection of 24 Japanese cicada species revealedthat while all species possessed Sulcia, only nine species retainedHodgkinia, and their genomes exhibited substantial structural instability.The remaining 15 species lacked Hodgkinia and instead harbored yeast-like fungal symbionts. Detailed phylogenetic analyses uncoveredrepeated Hodgkinia-fungus and fungus-fungus replacements incicadas. The fungal symbionts were phylogenetically intermingledwith cicada-parasitizing Ophiocordyceps fungi, identifying ento-mopathogenic origins of the fungal symbionts. Most fungal sym-bionts of cicadas were uncultivable, but the fungal symbiont ofMeimuna opaliferawas cultivable, possibly because it is at an earlystage of fungal symbiont replacement. Genome sequencing of thefungal symbiont revealed its metabolic versatility, presumably capableof synthesizing almost all amino acids, vitamins, and other metab-olites, which is more than sufficient to compensate for the Hodgkinialoss. These findings highlight a straightforward ecological and evo-lutionary connection between parasitism and symbiosis, which mayprovide an evolutionary trajectory to renovate deteriorated ancientsymbiosis via pathogen domestication.

cicadas | Ophiocordyceps | parasitic fungi | symbiotic fungi |symbiont replacement

Diverse insects are symbiotically associated with diverse mi-crobes (1–3). In particular, extremely intimate relationships

are found among host-symbiont associations underpinningstringent ecological and physiological necessities for energy,metabolites, or nutrients. For example, the majority of plant-sucking insects of the order Hemiptera, including aphids, white-flies, scale insects, psyllids, cicadas, spittlebugs, leafhoppers,planthoppers, and many others, are obligatorily dependent onsymbiotic microorganisms for provisioning of essential aminoacids and other nutrients that are deficient in their sole foodsource of plant sap (4–7). In most cases, the hosts have de-veloped specialized cells and organs, called bacteriocytes andbacteriomes, to which their specific symbionts are localized (8,9). In the maternal body, the symbionts migrate to developingoocytes, thereby ensuring vertical symbiont transmission throughhost generations (10, 11). In many cases, the symbiont phylogenymirrors the host phylogeny, indicating strict host-symbiontcospeciation over evolutionary time, which may exceed 100–200 million y (12, 13).

Notably, such intimate host-symbiont associations certainlyentail stability and continuity on one hand, but, on the otherhand, theoretical and empirical studies have shown that suchhost-symbiont associations may potentially suffer instability andcollapse in the long run (14, 15). In obligate and long-lastingsymbiotic associations, the symbiont genomes tend to exhibitdrastic size reductions and massive gene losses, which are drivenby relaxed natural selection acting on many symbiont genes un-necessary for the symbiotic lifestyle, and also by accumulation ofdeleterious mutations due to genetic drift facilitated by strongpopulation bottlenecks and a paucity of horizontal gene acqui-sitions inherent in the obligate intrahost lifestyle (16–18). Someinsect symbiont genomes are extremely reduced to 0.2 Mb orsmaller in size, encode less than 200 genes, and so have genomeseven smaller than some organellar genomes (19–22). By accu-mulating numerous mutations that could potentially lead to ge-nomic malfunctioning and instability, such tiny-genome symbionts,

Significance

Cicadas are dependent on the essential bacterial symbionts Sulciaand Hodgkinia. The symbiont genomes are extremely streamlinedfor provisioning of essential amino acids and other nutrients. Insome cicada lineages, Hodgkinia genomes are fragmented intonumerous minicircles, which may represent a critical stage of ge-nomic erosion close to collapse.Whatwould happen subsequently?Our survey of the Japanese cicada diversity revealed that whileSulcia is conserved among all species, the majority of them havelost Hodgkinia and instead harbor yeast-like fungal associates. Thefungal symbionts are phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, indicating recurrent symbiontreplacements by entomopathogens in cicadas and providing in-sights into the mechanisms underlying the parasitism-symbiosisevolutionary continuum, compensation of symbiont genome ero-sion, and diversification of host-symbiont associations.

Author contributions: Y.M. and T.F. designed research; Y.M., M.M., P.Ł., M.T., and X.-Y.M.performed research; J.P.M. contributed new reagents/analytic tools; Y.M., M.M., P.Ł., D.V.,and J.P.M. analyzed data; and Y.M. and T.F. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Data deposition: The sequences reported in this paper have been deposited in the DNAData Bank Japan Read Archive, www.ddbj.nig.ac.jp (accession nos. LC370451–LC371030)and the GenBank database www.ncbi.nlm.nih.gov/genbank/ (accession nos. MG737715–MG737734, CP029009–CP029028, SAMN08930808–SAMN08930810, and SAMN08939728–SAMN08939730; BioProject nos. PRJNA450103, PRJNA450106, PRJNA450107,PRJNA450109–PRJNA450112, PRJNA450114–PRJNA450119, PRJNA450122–PRJNA450127,PRJNA450129, and PRJNA427071).1To whom correspondence may be addressed. Email: umatsuura@gmail.com or t-fukatsu@aist.go.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1803245115/-/DCSupplemental.

Published online June 11, 2018.

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and potentially their hosts, may be near the edge of extinction dueto genome erosion (14, 15). There are many examples, however, asin aphids (23–28), scale insects (29, 30), spittlebugs (31, 32),leafhoppers (33–37), planthoppers (38, 39), weevils (40, 41), lice(42, 43), and others (1, 44, 45), wherein an ancient and presumablydegraded bacterial symbiont with essential biological function hasbeen lost and replaced by totally different microbial associates.Whether the degenerative trend of symbiont genome evolution isrelevant to the symbiont losses, replacements, and diversification,and if so, how, is mostly unanswered but remains an intriguingissue of evolutionary biology.In this context, a relevant case of such symbiont genome de-

generation may be observed in the bacterial cosymbionts ofsinging cicadas, Sulcia and Hodgkinia. Sulcia has a small genomeof less than 0.3 Mb in size and comprises an ancient and con-served symbiont lineage, whose evolutionary origin dates back tothe common ancestor of the Auchenorrhyncha (cicadas, spittle-bugs, leafhoppers, planthoppers, etc.) as long as 260 million yago (13, 46). By contrast, Hodgkinia is restricted to cicadas, in-dicating a relatively younger evolutionary origin than Sulcia, butits genome is even more drastically reduced, typically smallerthan 0.15 Mb (47, 48). The Sulcia genome encodes biosyn-thetic pathway genes for most essential amino acids, while theHodgkinia genome complementarily retains genes for the es-sential amino acids histidine and methionine and the vitaminscobalamin and riboflavin, thereby jointly supporting the growthand survival of host cicadas feeding solely on nutritionally de-ficient plant xylem sap (48, 49). Notably, in some cicada lineages,Hodgkinia has evolved into complexes of distinct cellular line-ages with even more reduced but complementary genomes,which is interpreted as an unusual means of further genomicdegradation (50–53). In extreme cases, the symbiont genome isbroken down into an assemblage of dozens of minicircles, eachencoding only a few genes, which may be leading to some criticalstage of genomic instability (51–53). What, then, might be thefate of the Sulcia-Hodgkinia-cicada cosymbiotic association ifindeed the genome complexity observed in Hodgkinia is non-adaptive or even maladaptive for the symbiosis?Here, we report that frequent losses of Hodgkinia have cer-

tainly occurred in the natural cicada diversity. Our survey of 24Japanese cicada species revealed that the majority, 15 species,lack Hodgkinia infection. Hodgkinia losses are estimated to haveoccurred repeatedly, at least three times and likely more. Strik-ingly, all of the Hodgkinia-free cicada species are associated withyeast-like fungal symbionts, uncovering recurrent evolutionarytransitions from Sulcia-Hodgkinia-cicada symbiosis to Sulcia-fungus-cicada symbiosis. Phylogenetically, the fungal symbiontsof cicadas are intermingled with cicada-parasitizing Ophio-cordyceps fungi, identifying the evolutionary source of the fungalsymbionts as the fungal parasites of cicadas. These resultshighlight a straightforward evolutionary connection betweenparasitism and symbiosis, and unveil an evolutionary trajectory tocompensate for a deteriorating ancient bacterial symbiont bydomesticated entomopathogens.

Results and DiscussionGeneral Features of Symbiotic Organs in Japanese Cicadas. The su-perfamily Cicadoidea (Hemiptera: Auchenorrhyncha) includesover 3,000 species of large-sized, plant-sucking insects known assinging cicadas, and consists of two families (Cicadidae andTettigarctidae) and several subfamilies (54). From the JapaneseArchipelago, 1 family (Cicadidae), 2 subfamilies (Cicadinae andCicadettinae), 15 genera, and 35 species of cicadas have beendescribed (55), of which we collected adult insects of 24 speciesrepresenting 13 genera (SI Appendix, Table S1). In the abdom-inal body cavity, in addition to gonads, fat bodies, and an ali-mentary tract, voluminous tissue masses resembling grape bunches,colored white, pink or yellow, were consistently observed, whichrepresented the symbiotic organs, called the bacteriomes, of thecicadas (Fig. 1).

Endosymbiotic Microbiota in Japanese Cicadas. The bacteriomesand other tissues were dissected from the cicada samples andsubjected to PCR amplification/cloning/sequencing/detection ofthe bacterial 16S rRNA gene for all 24 species representing73 populations and 219 individuals (SI Appendix, Table S1).Among them, dissected bacteriomes, often associated with fatbody fragments, from 20 samples representing 20 species weresubjected to metagenomic Illumina sequencing. In the meta-genomic assemblies, we identified mostly complete coding re-gions of mitochondrial genomes of host cicadas, complete Sulciagenome sequences, and genomic contigs of Hodgkinia and othermicrobial associates (SI Appendix, Fig. S1 and Tables S2–S5).

Fig. 1. Dissected symbiotic organs of cicadas. (A–C) Me. opalifera. (D–F)Tanna japonensis. (G–I) G. nigrofuscata. (J–L) P. kaempferi. (M–O) Auritibicenbihamatus. (P–R) C. facialis. (S–U) Mo. minuta. (Left) Photographs of adultfemales. (Center) Photographs show dissected abdominal organs. (Right)Photographs are enlarged images of dissected bacteriomes. bc, bacteriome;fb, fat body; gt, gut; ov, ovary.

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While a substantial proportion of metagenomic reads and scaffoldscorresponded to the nuclear genomes of the host cicadas, these datawere not analyzed further because of the very low genomic cover-age. We consistently identified 16S rRNA gene sequences of Sulciafrom all 24 Japanese cicada species, which were phylogeneticallyplaced in the cluster of cicada-associated Sulcia symbionts in theFlavobacteriaceae (SI Appendix, Fig. S2). On the other hand, al-though previous studies had identified Hodgkinia as another bac-teriome symbiont in North American, South American, andAustralian cicadas (47–53), our extensive PCR and metagenomicsurveys detected Hodgkinia from only nine of 24 Japanese cicadaspecies: three Platypleura species, three Auritibicen species, Kosemiayezoensis, Vagitanus terminalis, and Muda kuroiwae (SI Appendix,Fig. S3 and Tables S1, S2, and S5). In some cicada species, sec-ondary bacterial symbionts, including Wolbachia, Arsenophonus,Sodalis, and Spiroplasma, were also detected (SI Appendix, Fig.S1 and Tables S1 and S2).

Genomics of Sulcia and Hodgkinia of Japanese Cicadas. All 20 Sulciagenomes determined by metagenomic sequencing were of theexpected size, ranging from 0.24 to 0.28 Mb; were mostly syn-tenic with previously published Sulcia genomes; and encoded aset of bacterial genes similar to those identified in previouslyreported Sulcia genomes, which included most genes needed forsynthesizing essential amino acids (SI Appendix, Fig. S4 andTable S4). The phylogeny of Sulcia genome sequences was highlycongruent with the phylogeny of host mitochondrial genomesequences (SI Appendix, Fig. S5), confirming the expected codi-

versification between Sulcia and host cicadas over evolutionary time(13, 14). On the other hand, in all of the six Hodgkinia-associatedJapanese cicada species subjected to metagenomic sequencing, theHodgkinia-derived genomic contigs were never fully assembled, andtheir size, organization, guanine-cytosine (GC) content, and cover-age variability suggested their origins from different Hodgkinia ge-nomes coexisting in the same insect (SI Appendix, Figs. S1 andS6 and Table S5), as observed in some American cicadas (50–53). Inall six cases, the total size of the identified Hodgkinia genomiccontigs was greater, and much greater in some cases, than the size ofthe nonfragmented Hodgkinia genome identified from the NorthAmerican cicada Diceroprocta semicincta (48) (SI Appendix, TableS5). In five of the six species, we identified two or more distinctcopies of 16S rRNA genes, and in three of the six species, weidentified multiple copies of a conserved Hodgkinia protein-codinggene, rpoB (SI Appendix, Fig. S6), as observed in the South Amer-ican cicada genus Tettigades (50, 52). These observations stronglysuggested that the Hodgkinia genomes are also fragmented anddegenerated in Japanese cicadas. These Hodgkinia genomes wereleft as draft genome assemblies due to their complexity.

Conserved Sulcia and Frequent Lack of Hodgkinia in Japanese Cicadas.These results uncovered that while the ancient bacteriomesymbiont Sulcia is highly conserved, the bacteriome cosymbiontHodgkinia was missing in the majority of the Japanese cicadaspecies. This finding was striking in that the Hodgkinia genomeencodes biosynthetic pathways for several essential nutrients,including histidine, methionine, cobalamin, and riboflavin, which

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Fig. 2. In vivo localization and fine structure of Sulcia, Hodgkinia, and yeast-like fungal symbiont (YLS) of cicadas. (A–D) P. kaempferi. (E–H)Me. opalifera. (I–L) G. nigrofuscata. (A, E, and I) Whole-mount in situ hybridization of dissected bacteriomes. (B, F, and J) In situ hybridization of Technovit thin sections ofbacteriome units. (C, G, and K) In situ hybridization of Technovit thin sections of fat body cells. Blue, magenta, green, and yellow visualize DNA, Sulcia,Hodgkinia, and YLS, respectively. Insets (G′ and J′) are enlarged light microscopic images of YLS cells. bc, bacteriome; fb, fat body; tr, trachea. (D, H, and L)Transmission electron microscopic images of the microbial symbionts. Insets (H′ and L′) are enlarged images of YLS cells. cw, cell wall of YLS; H, Hodgkinia; lg,lipid granule; mt, mitochondrion of YLS; N, nucleus of host insect; n, nucleus of YLS; S, Sulcia; v, vacuole of YLS; Y, YLS.

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are absent from the Sulcia genome, and thus the metaboliccomplementarity between Sulcia and Hodgkinia has been pre-sumed to be important for survival of the cicadas feeding solely

Fig. 3. Light microscopic images of yeast-like symbiont cells released fromdissected cicadas. (A) G. nigrofuscata. (B) H. maculaticollis. (C) C. facialis. (D)Cryptotympana atrata. (E) Me. opalifera. (F) Meimuna kuroiwae. (G) Meimunaoshimensis. (H) Euterpnosia okinawana. (I) Tanna japonensis. (J) Mo. minuta.

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Fig. 4. Localization of Sulcia, Hodgkinia and yeast-like fungal symbiont atthe posterior pole of developing oocytes of cicadas visualized by in situhybridization. (A) P. kaempferi. (B) Me. opalifera. (C) G. nigrofuscata. Blue,magenta, green, and yellow indicate DNA, Sulcia, Hodgkinia, and yeast-likesymbiont (YLS), respectively. In B and C, YLS cells are seen in the symbiontball and also in the epithelial plug, which YLS was reported to infect forvertical transmission in planthoppers (78). ep, epithelial plug; fc, follicle cell;sb, symbiont ball.

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on nutritionally deficient plant xylem fluid (48, 49). How arethese cicadas capable of surviving without Hodgkinia? In an at-tempt to address this question, we carefully inspected the Japa-nese cicadas morphologically, histologically, and cytologically.

Detection of Vertically Transmitted Fungal Symbionts in CicadasLacking Hodgkinia. In the cicada species associated with bothSulcia and Hodgkinia, such as Platypleura kaempferi and Auriti-bicen japonicus, each bacteriome unit consisted of three cellularcomponents: surface sheath cells constituting the outermostepithelial cell layer to encase the whole bacteriome unit, pe-ripheral bacteriocytes comprising the surface layer beneath thesheath cells, and a central syncytial cytoplasm located at thecenter of the bacteriome unit (SI Appendix, Fig. S7 A and B andTable S6). Light microscopy, fluorescence in situ hybridizationtargeting bacterial 16S rRNA, and transmission electron mi-croscopy identified Sulcia in the peripheral bacteriocytes andHodgkinia in the central syncytial cytoplasm, respectively (Fig. 2A–D and SI Appendix, Fig. S8 A and B). On the other hand, inthe cicada species associated with Sulcia only, such as Meimunaopalifera, Graptopsaltria nigrofuscata, Cryptotympana facialis,Hyalessa maculaticollis, and Mogannia minuta, while the surfacesheath cells were clearly recognizable, the peripheral bacter-iocytes and the central cytoplasm were indiscernible and com-prised the inner bacteriome region (SI Appendix, Fig. S7 B–E andTable S6), where Sulcia was specifically localized (Fig. 2 E, F, I,and J and SI Appendix, Fig. S8 C, F, and I). Notably, when thesecicada samples were dissected, numerous yeast-like buddingparticles were observed under the light microscope (Fig. 3). PCRamplification and sequencing identified fungal 18S rRNA genesequences from these cicada species (SI Appendix, Table S1),

which exhibited the highest similarities to 18S rRNA gene se-quences of entomoparasitic fungi of the genus Ophiocordyceps,including Ophiocordyceps longissima (KJ878925), Ophiocordycepssobolifera (EF468972), and Ophiocordyceps yakusimensis(AB044632). Reexamination of the Illumina reads confirmed thepresence of fungal gene assemblies (SI Appendix, Fig. S1 andTable S2), although coverage values for the fungal assemblieswere generally low, which was likely due to the low efficiency ofDNA extraction from fungal cells with a thick cell wall. Fluo-rescence in situ hybridization targeting fungal 18S rRNA andtransmission electron microscopy visualized the yeast-like sym-bionts in the fat body surrounding the bacteriomes (e.g., Me.opalifera, C. facialis, Mo. minuta) (Fig. 2 G and H and SI Ap-pendix, Fig. S8 D and J), in the well-developed surface sheathcells (e.g., G. nigrofuscata) (Fig. 2 I, J, and L), or in both (e.g., H.maculaticollis) (SI Appendix, Fig. S8 F and G). Transmissionelectron microscopy confirmed that the fine structure of theyeast-like symbionts was typical of unicellular fungi with a nu-cleus, mitochondria, and thick cell wall (Fig. 2 H and L). Fluo-rescence in situ hybridization of ovaries dissected from adultfemales detected specific localization of not only Sulcia but alsothe yeast-like symbionts in developing oocytes, where the coin-fecting symbionts formed a ball-shaped mass at the posteriorpole (Fig. 4), indicating a vertical transmission route for theyeast-like symbiont that may be functionally equivalent to thetransmission of Sulcia and Hodgkinia.

Recurrent Losses of Hodgkinia and Replacements by FungalSymbionts. These results unveiled that while the ancient bacter-iome symbiont Sulcia has been stably maintained in cicadas, thebacteriome cosymbiont Hodgkinia has not, which may be related

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Fig. 5. Phylogenetic relationship of cicadas andtheir infection status with microbial symbionts. Amaximum-likelihood phylogeny inferred from 15 mi-tochondrial gene sequences and 22 tRNAs (14,733aligned nucleotide sites) of 20 Japanese cicada species,together with four previously studied American species(highlighted by #), is shown. Bootstrap support values areindicated on each node in the order of maximum-likelihood/neighbor-joining. Detected microbial symbi-onts are mapped on the right side of each species namewith orange circles for Sulcia, green circles for Hodgkinia,and yellow circles for the yeast-like fungal symbiont. Inthe green circles of Hodgkinia, the number 1, 2, or 4 in-dicates the number of distinct Hodgkinia genomes thatform a complex. C indicates highly fragmentedHodgkiniacomplexes in which the exact number of genomescould not be determined (51, 53). Colored triangles onthe phylogeny indicate the estimated replacement eventsfrom Hodgkinia to the fungal symbionts. Selectedimages of adult cicadas are depicted to the right of themaximum-likelihood phylogeny.A. bihamatus,Auritibicenbihamatus; C. atrata, Cryptotympana atrata; E. chibensis,Euterpnosia chibensis; G. bimaculata, Graptopsaltria bima-culata; Ma. tredecim, Magicicada tredecim; Me. iwasakii,Meimuna iwasakii; Me. oshimensis, Meimuna oshimensis;Ta. japonensis, Tanna japonensis; Te. nigricosta, Terpnosianigricosta; Te. vacua, Terpnosia vacua; Tet. ulnaria, Tetti-gades ulnaria; Tet. undata, Tettigades undata.

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to the extreme genome degeneration and fragmentation ob-served in some Hodgkinia lineages (50–53). On the grounds thatthe Hodgkinia-free cicada species always possess the fungalassociates, the evolutionary process must have entailed re-placement of Hodgkinia by the fungal symbiont. In this study, wefound no cicada individuals containing both Hodgkinia and thefungal symbiont (SI Appendix, Table S1). According to the phy-logeny of the Japanese cicadas based on mitochondrial genomesequences, on which their microbial symbionts were mapped,Hodgkinia has been replaced by the fungal symbiont repeatedly, atleast three times and possibly more (Fig. 5).

Phylogenetic Placement and Diversity of Fungal Symbionts in Cicadas.Molecular phylogenetic analysis based on fungal 18S rRNA genesequences showed that the fungal symbionts of cicadas formed arelatively well-supported clade within the genus Ophiocordyceps(SI Appendix, Fig. S9), an ascomycetous group consisting ofentomopathogenic fungi with a number of cicada-parasitizingspecies (56, 57). This phylogenetic pattern highlighted a close

evolutionary connection between the fungal symbionts of cicadasand the Ophiocordyceps entomopathogens. Furthermore,four additional fungal nuclear genes were amplified by PCR andsequenced for all of the 15 fungus-associated cicada species(SI Appendix, Table S1), which yielded a better resolvedphylogenetic relationship of the fungal symbionts (SI Appendix,Fig. S10). Phylogenetic comparison of the host cicadas and thefungal symbionts (Fig. 6) showed that several cicada-parasitizingfungi, such as O. longissima, O. yakusimensis, and O. sobolifera, wereplaced within or just outside the clade of the cicada symbionts,favoring the hypothesis that the fungal symbionts of cicadas haveevolved from cicada-parasitizing Ophiocordyceps fungi.Phylogenetic comparison of the host cicadas and the fungal

symbionts also showed that the phylogenetic relationship of thecicada symbionts was locally concordant with the phylogeneticlineages of the host cicadas, as exemplified by the fungal symbiontlineages associated with Graptopsaltria spp., Cryptotympana spp.,and Meimuna spp. (except Me. opalifera), which was indicative of

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Fig. 6. Cophylogenetic analysis of host cicadas and their yeast-like fungal symbionts (YLS). (A) Maximum-likelihood phylogeny of 20 Japanese cicada speciesinferred from mitochondrial genome sequences (15 genes and 22 tRNAs, 14,733 aligned nucleotide sites), with two South American cicada species, Tettigadesspp., as outgroup taxa. Fungus-associated cicada species are shown in black, whereas Hodgkinia-harboring cicada species are shown in gray. (B) Maximum-likelihood phylogeny of their fungal symbionts based on five nuclear gene sequences (4,392 aligned nucleotide sites), with allied Ophiocordyceps ento-mopathogenic fungi as ingroup and outgroup taxa. Host-symbiont connections are shown by black dashed lines. Cicada-parasitizing Ophiocordyceps fungiallied to the cicada symbionts, namely, O. yakusimensis, O. longissima, O. sobolifera, and Ophiocordyceps heteropoda, are highlighted by colors, and theirhost range records are shown by colored dotted lines according to ref. 57. Estimated replacement events from Hodgkinia to a fungus or from a fungus toanother fungus are mapped on the phylogeny. A. bihamatus, Auritibicen bihamatus; C. atrata, Cryptotympana atrata; E. chibensis, Euterpnosia chibensis;G. bimaculata, Graptopsaltria bimaculata; Me. iwasakii, Meimuna iwasakii; Me. kuroiwae, Meimuna kuroiwae; Me. oshimensis, Meimuna oshimensis;O. brunneipunctata, Ophiocordyceps brunneipunctata; Ta. japonensis, Tanna japonensis; Te. nigricosta, Terpnosia nigricosta; Te. vacua, Terpnosia vacua.

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some degree of host specificity, stable vertical transmission, andhost-symbiont codiversification.Globally, however, the phylogeny of the fungal symbionts was

incongruent with the phylogeny of the host cicadas, reflectingdynamic evolutionary trajectories of the fungal symbionts overdeeper evolutionary time, presumably involving repeated acquisi-tions, losses, and replacements. In Fig. 6, three replacement eventsfrom Hodgkinia to a fungus and three replacement events from afungus to another fungus are estimated and mapped. It should benoted, however, that this estimate is parsimonious and minimal innumber and that the actual evolutionary process may be morecomplex and dynamic. It should also be kept in mind that althoughseemingly less likely, the possibility of evolutionary reversals froma symbiotic to parasitic lifestyle cannot be excluded.

Recurrent Evolution of Fungal Symbionts from Parasitic Fungi inCicadas. These results strongly suggest that the fungal symbi-onts of cicadas have repeatedly evolved from cicada-parasitizingOphiocordyceps fungi, highlighting a straightforward connectionbetween parasitism and symbiosis. It seems plausible, althoughspeculative, that the ecological overlap between the cicada nymphsand the Ophiocordyceps entomopathogens in the plant rhizosphere(58), in combination with the ability of the Ophiocordyceps-alliedentomopathogens to evade the insect immunity and survive andproliferate inside the insect body cavity (59–61), might havepredisposed the recurrent evolution of the fungal symbionts fromthe fungal parasites in cicadas. In this context, it is notable thatyeast-like fungal symbionts have been reported from diverse in-

sect groups (1, 62–64), and some of them were identified to bephylogenetically allied to Ophiocordyceps entomopathogens as inaphids (25, 65, 66), scale insects (29), planthoppers (38, 39, 67–69), and leafhoppers (33, 35, 36, 70), suggesting the possibilitythat the Ophiocordyceps entomopathogens might be serving as anenvironmental source for the evolution of novel fungal symbiontsin diverse insects.

Cultivation of Fungal Symbiont of Cicadas. We attempted to culti-vate the fungal symbionts from the fungus-associated Japanesecicadas representing 6 species, 11 populations, and 53 individualson standard nutrient agar media (SI Appendix, Table S7). Frommost of the samples, no growing Ophiocordyceps fungi wereobtained, except for occasional fungal contaminants that wereverified with 18S rRNA gene/internal transcribed spacer (ITS)region sequencing. Notably, numerous fungal colonies of uniformmorphotype were reproducibly isolated only from Me. opalifera(Fig. 7 and SI Appendix, Table S7). Three fungal strains isolatedfrom adult cicadas collected at three distinct localities in Japanyielded almost identical 18S rRNA gene sequences to each otherand also to the fungal symbiont sequences derived from dissectedbacteriomes of Me. opalifera (SI Appendix, Fig. S9), indicating thatthe fungal symbiont of Me. opalifera is cultivable. The symbiontcultivability in Me. opalifera may reflect the recent acquisition ofthe fungal symbiont in the host lineage, which is closely relatedto the cicada-parasitizing fungus O. longissima and derived froman allied cicada parasite (Fig. 6 and SI Appendix, Figs. S9 andS10). The cultivated strains of the fungal symbiont grew slowerthan the contaminant fungi that quickly grew hyphae and formedlarge colonies. After saline-suspended symbiont cells from adultMe. opalifera were spread on agar media, it took as long as over amonth at 25 °C, or 2–3 wk at 28 °C, to form small colonies ofseveral millimeters in diameter consisting of radial hyphae (Fig. 7A and B). Subsequently, the colonies became thicker and mound-shaped, rather than spreading flat, thereby constituting a dense,thick, and hard mycelial mass with a layered structure, whichlooked like the fungal sclerotium (Fig. 7 C–I). It is notable thatupon infection and killing of their insect host, Ophiocordycepsentomopathogens fill up the host body with a hardened mycelialmass called the sclerotium, and finally form fruiting bodies toproduce ascospores and/or conidia (56, 57). The cultivable fungalsymbiont ofMe. opalifera, which exhibits prevalent infection in hostpopulations (SI Appendix, Tables S1 and S7) and vertical trans-mission to developing oocytes (Fig. 4B), seems like an intermediatestatus between the free-living Ophiocordyceps entomopathogensand the uncultivable fungal symbionts associated with other cicadalineages. It may provide a promising model system for gaining in-sights into how the evolutionary transition from free-living throughcultivable to uncultivable fungal associates has proceeded, as re-cently highlighted in gut bacterial symbioses in stinkbugs (71–74).Whether the cultivable fungal symbiont is detectable, existing, andsurviving in the habitats ofMe. opalifera is of ecological interest anddeserving of future field surveys.

Genomic Features of Fungal Symbiont: Insight into MetabolicComplementarity and Symbiont Replacement. The fungal symbi-ont of Me. opalifera was grown in liquid culture for preparationof genomic DNA of sufficient purity and quantity suitable forPacBio single-molecule genome sequencing. Sequencing on foursingle-molecule real-time (SMRT) cells resulted in 186-fold cov-erage of a draft genome, which was 25.1 Mb in size and assembledinto 32 contigs. Subsequent analyses revealed a highly compactgenome with a 60.4% GC content and ∼7,000 protein-codinggenes (0.278 genes per kilobase) with a median gene lengthof 1,580 bp (median exon size of 316 bp, median intron size of57 bp). Repetitive DNA sequences made up only 9.55% of theassembled length, the majority of which were simple repeatsand LTR elements (4.04% and 3.88%, respectively). We iden-tified 14 full-length ribosomal RNA operons as well as a singlemitochondrial genome contig of 170 kb in size (SI Appendix, TableS8). The fungal symbiont genome of Me. opalifera retained all

A B

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Fig. 7. Cultivated fungal symbiont strain from the cicada Me. opalifera.(A) Small colonies on a peptone-supplemented potato dextrose agar platearound 1 mo after inoculation. (B) Enlarged image of the colonies, whichconsist of radially arranged hyphae. (C) Large colonies about 3 mo after in-oculation. Note that the colonies are thick and hard, constituting a dense massof mycelia. (D) Mound-shaped colonies whose inoculum was a small piece ofthe dense mycelial mass cut from the precultured symbiont colony. Note thatthe colonies grow as hard and coherent fungal masses rather than stretchinghyphae into flat colonies. (E) Scanning electron microscopic image of a cutplane of the mound-shaped colony, in which layered structures (layer I to layerIV from surface to inside) are observed. (F) Layer I, consisting of filamentoushyphae arranged outward like a rug surface. (G) Layer II, consisting of denselypacked hyphae. (H) Layer III, consisting of entangled filamentous hyphae.(I) Layer IV, consisting of a matrix filling the space between relatively sparsefilamentous hyphae.

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synthesis pathway genes for essential and nonessential aminoacids, B vitamins, and nitrogen recycling, which included thesynthesis pathway genes for histidine and methionine that areprovisioned by Hodgkinia (SI Appendix, Fig. S11). These resultshighlight metabolic versatility of the fungal symbiont that ismore than sufficient to compensate for the absence of Hodgki-nia. Genome sequencing and comparative genomics of the fungalsymbionts of other cicada lineages will provide further insights intothe processes and mechanisms of the dramatic symbiont replace-ments in cicadas.

Ecological and Evolutionary Connection of Fungal Symbionts andParasitic Fungi. With all these results taken together, we pro-pose a hypothetical perspective as to how the fungal symbionts ofcicadas and the cicada-parasitizing Ophiocordyceps entomo-pathogens are interconnected to each other ecologically andevolutionarily in the natural environment (Fig. 8). Cicada nymphsspend many years in the soil of the plant rhizosphere, where theyfeed solely on xylem fluid from plant roots (55, 75). The ecology ofcicada nymphs with constant and long-lasting exposure to humidand microbe-rich soil seems to facilitate contact and infection withpathogenic microorganisms. Notably, cicada parasites occupy asubstantial fraction of the diversity of Ophiocordyceps-allied ento-mopathogens: For example, of some 240 species described fromJapan, over 30 species (∼13%) were reported to exploit cicadas(57). Hence, it is expected that cicada nymphs are frequently andconstantly challenged by such fungal parasites in the natural envi-ronment. Upon invasion into the body cavity and before killing theirinsect host, Ophiocordyceps-allied entomopathogens proliferate inthe hemolymph as nonhyphal yeast-like cells called hyphal bodies orblastospores (61, 76, 77). Because the morphology and localizationof the hyphal bodies are quite reminiscent of those of the fungalsymbionts, although speculative, we suggest the possibility that theyeast-like fungal symbionts may be derived from, and evolutionarily

homologous to, the hyphal bodies of the fungal parasites. Oncesome variants/mutants of the fungal parasites attenuate virulenceand become benign, it is expected that such fungal mutants mayestablish nonlethal and chronic infection within the host body in theform of hyphal bodies, instead of killing the host and developingfruiting bodies. However, it should be noted that evolving themechanisms for getting entry into developing oocytes in the ma-ternal body may be a substantial obstacle to establishing a trans-generational association via vertical transmission (78, 79).Considering the general metabolic versatility of fungi capableof synthesizing amino acids, vitamins, and other nutrients, suchchronic fungal infections may entail a fitness benefit, especially incicada lineages whose Hodgkinia has suffered massive genome de-generation. Furthermore, such fungal infections may additionallyentail nonnutritional fitness benefits for the host cicadas, like con-ferring resistance to further microbial infections (80–82). Pre-sumably, such fungal infections, establishments, and replacementsare ongoing in the plant rhizosphere, which may have driven therecurrent evolution of the fungal symbionts in place of the ancientbacterial symbiont lineage. The ecological and evolutionary con-nection of the fungal symbionts to the fungal parasites in cicadasprovides an impressive example of the parasitism-mutualismevolutionary continuum that has been advocated theoretically(83–87). In this context, the possibility that some fungal symbiontsmight exhibit a dual transmission strategy, in which verticaltransmission to eggs in reproducing females coexists with hostkilling and spore formation for horizontal transmission in post-reproduction females and/or males, is theoretically predicted,whose verification deserves future studies.

On Diversity of Cicadas, Ophiocordyceps Entomopathogens, and FungalSymbionts. Thus far, over 3,000 species of cicadas and some 500species of Ophiocordyceps-allied entomopathogenic fungi havebeen described (54, 57). However, the taxonomy and systematics of

hatchling

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Fig. 8. Schematic illustration of the hypotheticalecological and evolutionary scenario as to how thefungal symbionts have been recruited from fungalparasites in cicadas.

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these groups are far from complete, and a large number of speciesare still waiting for discovery and description. Both cicadas andOphiocordiceps fungi are the most diversified in warm and humidtropical/subtropical regions in the world, where the biodiversityis enormous, but thorough surveys are limited (54, 57). Recentstudies on microbial symbionts of cicadas have identified thebacterial symbionts Sulcia and Hodgkinia but failed to detectthe fungal symbionts (13, 47, 48, 50–53, 88). We expect thatfuture studies on the diversity of tropical cicadas will uncovermany more dynamic aspects of the evolution of microbialsymbionts, plausibly involving numerous acquisitions, losses,and replacements across bacterial and fungal associates. Con-sidering the metabolic versatility of the fungal symbiont relativeto bacterial symbionts, fungal replacement of both Sulcia andHodgkinia might also be possible in cicadas, as reported in someplanthoppers and leafhoppers, wherein Sulcia and other ancientbacterial symbionts have been completely lost and taken over byfungal associates (35, 38). In the classic extensive histological sur-veys by German microbiologists (1, 44, 45, 89, 90), a comprehensivestudy detected fungal symbionts in as many as 237 (64%) of 370species of plant-sucking hemipteran insects representing theAuchenorrhyncha (cicadas, spittlebugs, leafhoppers, treehoppers,planthoppers, etc.) (44), and recent studies, including this study,have shown that some of them are Ophiocordyceps-allied fungalsymbionts (33, 35, 37–39, 67, 70). Here, we point out that suchdynamic symbiont recruitment from fungal parasites may be takingplace in diverse insects more generally than previously envisaged.

Concluding Remark. In North America, cicadas are well known bythe general public for their relatively large size, their loud andmusical songs, and their massive periodical emergence from theunderground (75). In Asia, people recognized that bizarre-shaped mushrooms sometimes grow out of cicada nymphs andother insects underground, developing the mystic notion of an-imal/plant transformation and utilizing the insect/fungus com-plex for traditional medicinal purposes (91, 92). In Europe, earlymicrobiologists microscopically described the universal occur-rence of not only bacterial symbionts but also yeast-like fungalsymbionts in a variety of insects (1, 44, 45, 89, 90), although theirmicrobiological identity has long been elusive due to their fas-tidious nature and the lack of molecular tools at that time.Mycologists have described Ophiocordyceps and allied fungi asinsect parasites, including many cicada-parasitizing species (56,93–96). Recent molecular phylogenetic approaches have identi-fied some of the yeast-like fungal symbionts of insects as closerelatives of the Ophiocordyceps entomopathogens (25, 33, 35, 37–39, 65–70). Recent genomic approaches to the insect-associated

microbial communities have uncovered many striking cases ofdrastic size reduction and extreme metabolic streamlining inancient bacterial symbiont genomes, some of which look likethey are almost going beyond the limit of being able to sustainbasic cellular functioning (19–22). Among them, the ancientbacterial symbiont of cicadas, Hodgkinia, represents a strikingcase: The genome is reduced to only a small percentage of thesize of the Escherichia coli genome, encodes less than 200 genes,supplies only a few essential nutrients to the host cicada, and isoften highly fragmented into a number of minicircles, which isindicative of genomic instability and possibly at the edge of ex-tinction due to genome erosion (47, 48, 50–53). In this study,these divergent lines of previous knowledge on cicadas and theirassociated microbes across time, space, and scale are integrated intoa coherent picture, which sheds light on the dynamic ecological andevolutionary aspects of endosymbiosis entailing continual birth,decline, collapse, and renewal of intimate host-symbiont associations.

Materials and MethodsCicada samples used in this study are listed in SI Appendix, Table S1. PCR,cloning, and sequencing of bacterial and fungal genes from dissected cicadatissues were performed using the primers listed in SI Appendix, Table S9.Metagenomic libraries of dissected symbiotic organs were constructed usingthe TruSeq DNA PCR-Free Library Preparation kit or the NEBNext Ultra DNALibrary Prep Kit, and sequenced on an Illumina HiSeq 2500 system. Quality-trimmed reads were assembled, and resultant contigs were annotated andvisualized using custom Python and Processing scripts. Bacterial and fungalsymbionts in cicada tissues and cells were visualized and observed by lightmicroscopy, whole-mount fluorescence in situ hybridization, in situ hybrid-ization of methacrylate resin thin sections, and transmission electronmicroscopy. In situ hybridization was performed using the fluorochrome-labeled probes listed in SI Appendix, Table S10. Fungal cultivation wasconducted using nutrient agar media supplemented with antibiotics as de-tailed in SI Appendix, Table S7.

Complete details on the materials and methods are provided in SI Ap-pendix, SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Junko Makino, Koki Murano, KoheiOguchi, Keisuke Shimada, Masami Hayashi, Masaaki Kimura, Nahomi Kaiwa,Takahiro Hosokawa, Takashi Kanbe, and Yoshiko Ishii for cicada samples;Masami Hayashi and Mamoru Yasuda for cicada and fungal photographs;Chiaki Matsuura for cicada illustrations; and Yoshitomo Kikuchi for logisticsupport. This study was supported by Japan Society for the Promotion ofScience (JSPS) KAKENHI Grants JP26840116 (to Y.M.) and JP25221107 andJP17H06388 (to T.F.); by a general research grant of the Institute of Fermen-tation, Osaka; by National Science Foundation Grants IOS-1256680, IOS-1553529, DGE-1313190, IAA-1443108, and EPS-1101342; and by NASA Astro-biology Institute Award NNA15BB04A. Y.M., M.M., and M.T. were supportedby a JSPS Fellowship for Young Scientists.

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